Abstract: :
Purpose: In the rd mouse, photoreceptor degeneration is dueto a mutation of the rod–specific enzyme cGMP phosphodiesterase,resulting in permanently opened cGMP–gated cation channelsin the rod outer segment membrane, allowing Na+ and Ca2+ ionsto enter the cell, resulting in toxic levels of Ca2+. To identifypathways involved in the Ca2+–induced cell death of therd rods, we evaluated gene expression (GE) in the rd and wildtype (wt) mouse retina over the known time course of degeneration,and assessed simultaneous morphological changes with in vivoimaging.Methods: Total RNA was used to generate ds–cDNA, whichserved as a template to transcribe biotinylated cRNA. The labeled,fragmented probes were hybridized to U74A oligonucleatide arrays(Affymetrix). The raw data (PM–values) was normalizedaccording to an algorithm developed to integrate binding interactionsof probe sequences on microarrays (Zhang et al., 2003). Toolsfor hierarchical, functional and gene ontology (GO) term clusteringwere implemented in Matlab Statistical Toolbox functions. Selectedgenes were verified at the mRNA level using the QuantiTect SyberGreen PCR Kit, and at the protein level using immunohistochemistry.Morphological in vivo data were obtained using a scanning laserophthalmoscope (Heidelberg Engineering HRA).Results: (A) 182 genes passed the selection criteria (low standarddeviation and high correlation between samples), falling intosix clusters. For any given pair of genes, an expression profilecorrelation distance and a semantic distance (one for each classof GO terms) was established. (B) GE in rd started to deviatefrom wt by P10, with a reduction in photoreceptor– andan increase in apoptosis– and neuroinflammation–specificgenes. (C) Quantitative RT–PCR and immunohistochemistryconfirmed the increase in neuroinflammatory processes. (D) Persistentvitreal vessels and reduced retinal capillaries beyond P14 indicateda developmental delay. Between P14 and P21, an increase in retinaland vascular damage was observed.Conclusions: Our results suggest that photoreceptor degenerationin the rd mouse is a process starting with the primary insultof Ca2+–toxicity followed by a secondary insult involvingmulti–destructive pathways such as apoptosis and neuroinflammation,presumably boosting morphological changes. All of these componentsneed to be addressed if rods are to be successfully protected.